Dissecting the Functions of Conserved Prolines within

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Dissecting the Functions of Conserved Prolines within Transmembrane Helices of the D2 Dopamine Receptor Ethan B. Van Arnam,† Henry A. Lester,‡ and Dennis A. Dougherty*,† †

Division of Chemistry and Chemical Engineering and ‡Division of Biology, California Institute of Technology, Pasadena, California 91125, United States

bS Supporting Information ABSTRACT: G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing R-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis
trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P2105.50, a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures.

P

roline stands apart from the other 19 canonical amino acids. Its cyclic side chain uniquely shapes protein structure and facilitates protein dynamics. As proline’s side chain substantially restricts its backbone ψ angle and removes a backbone hydrogen bond donor, proline disrupts the regular structures of both R-helices and β-sheets and can introduce flexibility into these structures.1
4 Further, proline’s backbone N-substitution biases its peptide bonds to the cis conformer, relative to other peptide bonds. The cis
trans isomerization of proline peptide bonds is well documented to mediate protein folding and mature protein function.5 Here we examine which of the above properties contribute to function of the D2 dopamine G protein-coupled receptor (GPCR) at conserved proline sites within its transmembrane R-helices (TMs), using conventional and unnatural amino acid mutagenesis. Transmembrane proline residues are a characteristic feature of GPCRs and are found in five of the D2 receptor’s seven TMs. These prolines are also highly conserved; three (P2015.50, P3886.50, and P4237.50) are the most conserved residue of their respective helix among Class A GPCRs, and the remaining two (P892.59, P1694.59) are conserved among aminergic GPCRs. (Superscripts refer to Ballesteros
Weinstein numbering, in which the most conserved residue of helix X is denoted X.50).6 A wealth of conventional mutagenesis studies has already established that these prolines play some significant functional role in GPCRs.7
14 Proline kinks have long been hypothesized to mediate the helical movements involved in GPCR activation, acting as pivot points, hinges, and/or swivels to expose a G protein binding site at the intracellular end of the helical bundle.15,16 A recent crystal structure of the closely related D3 dopamine receptor r 2011 American Chemical Society

Figure 1. Two views of the crystal structure of the D3 dopamine receptor (PDB code 3PBL), highlighting the prolines considered here.15 Numbering is for the corresponding residues in the D2 receptor.

confirms that the prolines investigated here are associated with helical kinks, especially prominent in TMs 2, 6, and 7 (Figure 1).17 P1694.59 sits only one helical turn from the N-terminus of TM4, although the pre-proline turn is indeed kinked relative to the rest of the helix. Interestingly in the D3 and other GPCR crystal structures, TM5 is only slightly kinked and instead Received: May 13, 2011 Accepted: July 21, 2011 Published: July 21, 2011 1063

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as revealed by a series of cyclic proline analogues with varying cis preferences. Finally, we have characterized an important proline
aromatic interaction in the Cys loop of the muscle-type nicotinic acetylcholine receptor, in which the hydrophobicity of a Phe side chain preceding proline is important for receptor function.30 Using the various unnatural amino acids discussed above, we sought to determine which of these phenotypes is most applicable to the transmembrane prolines of the D2 receptor.

’ RESULTS AND DISCUSSION Figure 2. Hydrogen bonding patterns in an R-helix for (left to right) a typical amino acid; proline; an N-methyl amino acid; and an Rhydroxy acid.

possesses a prominent bulge in the helical turn preceding P2015.50.17
22 Proline’s ability to distort helices most obviously comes from its lack of backbone hydrogen bond donor ability, which frees its would-be hydrogen bond acceptor, the backbone carbonyl of the residue i
4 from proline (Figure 2). Additionally, the proline side chain introduces a steric clash with the i
4 carbonyl. The net effect is usually manifested as a kink, which often frees the carbonyl i
3 to proline as well.2,23,24 Since the 19 other canonical amino acids are ill-suited to probe the unique properties of proline, unnatural amino acid mutagenesis is an especially valuable tool to dissect the basis for a given proline’s functional importance. As structural information on GPCRs continues to accrue, the subtle and high precision probes provided by unnatural residues will become increasingly valuable. Tests of detailed structural environments and specific functional roles are significantly facilitated by the unnatural amino acid methodology. Here we compare the effect of replacing each of 5 TM prolines of the D2 receptor with unnatural R-hydroxy acids and an Nmethyl amino acid (Figure 2). We also consider cyclic proline analogues and conventional amino acids that correspond to the R-hydroxy acids. Unnatural R-hydroxy acids introduce a backbone ester that, like the proline peptide bond it replaces, is not a hydrogen bond donor (Figure 2). N-Me-Ala, effectively proline lacking its side chain Cγ, shares N-substitution (and thus lack of hydrogen bond donor ability) with proline but has greater conformational freedom. Finally, proline analogues that vary the size of the ring or introduce substituents can probe tolerance for subtle changes to the proline side chain as well as cis
trans isomerization. Pipecolic acid (Pip) and azetidine-2-carboxylic acid (Aze), six- and four-membered ring analogues of proline, respectively, have a greater cis bias relative to proline, whereas 2-methylproline (2-Me-Pro) is substantially more trans-biased.5 Previously, we have characterized three distinct phenotypes for functionally important proline residues in ligand-gated ion channels. In the M1 transmembrane helix of both the R subunit of the muscle-type nicotinic acetylcholine receptor and the 5-HT3A receptor, a conserved proline in the middle of the helix can be replaced by a range of R-hydroxy acids, giving essentially wild-type behavior.25,26 In contrast, incorporating any canonical amino acid other than proline gave a nonfunctional receptor. A second phenotype was seen in the M2
M3 loop of the 5-HT3A receptor.27 In this system, cis
trans isomerization of a proline peptide bond is critical for receptor gating (see also refs 28 and 29),

Experimental Approach. We utilized a recently optimized electrophysiological readout of the D2 receptor that yields quantitative dose
response relationships from receptor activation of coexpressed GIRK1/GIRK4 channels in Xenopus laevis oocytes and is amenable to unnatural amino acid mutagenesis by nonsense suppression.31 Here we probe five new sites of the D2 dopamine receptor with an array of unnatural residues, establishing the generality of this protocol for evaluating GPCR function. All unnatural amino acid and hydroxy acid mutations as well as the valine mutants noted in Table 1 were generated by nonsense suppression, whereas all other mutants were expressed conventionally. A wild-type rescue experiment (incorporating Pro by

Table 1. Conventional Amino Acid, Hydroxy Acid, and N-Me-Ala Mutationsa site

EC50 (nM)

wild-type Pro892.59

Pro1694.59

Pro2015.50

Pro3886.50

Pro4237.50

EC50 (nM)

39 Gly

35

N-Me-Ala

Ala

65

Aah

Valb

53

Vah

47

Leu

110

Lah

140

Gly

180

N-Me-Ala

40

Ala Valb

1300 1100

Aah Vah

59 76

Leu

>2000c

Lah

100

Gly

>2000c

N-Me-Ala

Ala

600

Aah

Val

>2000c

Vah

330

Leu

NDd

Lah

NDd

N-Me-Ala

46 140

17 180

Gly

67

Ala

230

Aah

92 58

Valb

>2000c

Vah

190

Leu

1000

Lah

35

N-Me-Ala

22

Gly

61

Ala

140

Aah

21

Valb Leu

170 NDd

Vah Lah

45 NDd

a

Reproducibility of the data is discussed in text. Errors for the goodness of fit to the Hill equation are presented in the Supporting Information. b Conventional mutant generated by nonsense suppression. c Attempts to measure large values of EC50 are compromised by background responses to dopamine at concentrations >100 μM. d No dopamineinduced current detected for dopamine concentrations up to 1 mM 1064

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Figure 3. Results for incorporating R-hydroxy acids, their amino acid analogues, Gly, and N-Me-Ala. At four sites, indicated by ≈, EC50 is too large to determine accurately. / = no current detected.

nonsense suppression) at each proline site yielded an EC50 within 2-fold of the wild-type value (see Supporting Information), validating the ability to perform nonsense suppression at all residues considered here. Because of the indirect nature of the assay, interpretation of EC50 requires some caution. A detailed discussion of this issue has been presented elsewhere.32 For present purposes, we consider EC50 values that differ by a factor of greater than 2 to be distinguishable in this assay. General Observations. We began our investigation of the D2 receptor’s transmembrane prolines by incorporating R-hydroxy acids at each site. For the most part, incorporation of an R-hydroxy residue is not strongly perturbing; all but one of the hydroxy acid mutants we could characterize gave EC50's within 5-fold that of wild-type (Figure 3, Table 1). In addition, each conventional amino acid residue is typically much more perturbing than its unnatural R-hydroxy acid analogue. This trend suggests the importance of lacking a hydrogen bond donor at these sites. N-Me-Ala provides an alternative way to remove the backbone NH group, and it is also well-tolerated at all sites. Given the results for R-hydroxy residues and N-Me-Ala, it is not surprising that various cyclic proline analogues, which are much more similar to proline, are well-tolerated at all transmembrane proline sites, yielding EC50's within 5-fold that of wild-type (Table 2). Mutations of proline to the conventional amino acids Ala, Val, and Leu range from modestly perturbing to dramatically perturbing (EC50 shifts of 3.5-fold to >2000-fold from wild-type), with the exception of P892.59, discussed below. Generally, Gly is less perturbing than the other natural amino acids, with the glaring exception of P2015.50G. These conventional mutants provide a useful reference to which we can compare the R-hydroxy mutants. The P892.59 site, despite >70% conservation among aminergic receptors, accepts diverse mutations with only modest functional

consequences. All mutations made, including conventional mutations, give wild-type EC50's or relatively small shifts from wildtype. Evidently this site, which is located in a helix not intimately involved in ligand binding or conformational changes, does not require the unique side chain and hydrogen bonding properties conferred by proline, and we will not consider it further here. P1694.59, P3886.50, and P4237.50. For the TM4, 6, and 7 proline sites, we observe hydroxy acid mutations (to Aah, Vah, or Lah; “ah” signifying R-hydroxy) in all cases to be less perturbing than the corresponding conventional amino acid mutations (to Ala, Val, or Leu) (Figure 3, Table 1). In fact, most of the hydroxy acid mutants for these sites (P1694.59Aah, P1694.59Vah, P3886.50Aah, P3886.50Lah, P4237.50Aah, and P4237.50Vah) have EC50's that are not meaningfully different from that of wild-type (i.e., within the margin of error we can expect from this assay). As hydroxy acids and proline both lack backbone hydrogen bond donors, this property alone may account for proline’s functional role at these sites. However, hydroxy acids do introduce additional perturbations; they weaken the hydrogen bond acceptor strength of the preceding residue’s backbone carbonyl and create an electrostatic repulsion between the introduced main chain oxygen and the i
4 carbonyl. An alternative way to remove the backbone NH of a peptide bond is with N-Me-Ala, which contains a fragment of the proline ring and does not significantly perturb the backbone carbonyl. At the TM4, 6, and 7 proline sites, N-Me-Ala is not largely perturbing, being essentially equivalent to Aah and producing EC50's within 2.5-fold that of wild-type (Figure 3, Table 1). This result suggests that the carbonyl mutation associated with R-hydroxy residues is not a large factor in these helices. Given the tolerance for R-hydroxy and N-methyl residues, it is not surprising that the proline analogues Aze and Pip are also well tolerated in TM4, 6, and 7 (Table 2). As described in previous work, Aze and Pip show different intrinsic cis
trans preferences than Pro.5 The minimal impact of these mutations indicates that cis
trans isomerization is not an essential component of receptor 1065

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Table 2. EC50 Values (nM) for Proline Analogue Mutationsa

site

Pip

Aze

2-Me-Pro 64

Pro892.59

26

26

Pro1694.59

19

25

37

Pro2015.50

34

41

160

Pro3886.50 Pro4237.50

82 80

47 15

130 130

a

Reproducibility of the data is discussed in text. Errors for the goodness of fit to the Hill equation are presented in the Supporting Information.

function. While varying ring size does not substantially alter function, introducing a methyl substituent at proline’s R carbon (2-Me-Pro) does produce measurable EC50 shifts at the 3886.50 and 4237.50 sites but not at 1694.59. Taken together, the mutagenesis results above indicate that the key function of proline at these sites is to disrupt the R-helix by removing the key backbone NH. Any strategy that accomplishes this, including cyclic residues, R-hydroxy residues, or N-methyl residues, produces receptors with essentially wild-type function. Interestingly, Gly is less perturbing than the other conventional amino acids at these sites (Figure 3, Table 1). Glycine is well-known to have an especially low propensity for helical structures relative to other amino acids (second-lowest only to proline),33,34 a property believed to stem both from its high conformational entropy and from its minimal burial of solventaccessible surface area in helices.35 The good tolerance of Gly at these sites suggests a generic functional requirement for a helix breaker at these sites. Proline appears to accomplish this by lack of a backbone hydrogen bond donor, but Gly can achieve the same end by different means. We do note that although the P4237.50G mutant gave a near-wild-type EC50, it produced signals that were generally small, suggesting either poor expression or diminished receptor efficacy, possibilities we cannot differentiate with our assay. Because the R-hydroxy residues do not seriously compromise receptor function at these sites (the way their R-amino analogues do), we can use the R-hydroxy data to consider the effects of side chain variation. The largest perturbations are seen for Lah at P1694.59 and Vah at P3886.50. This suggests that the 1694.59 site is sensitive to size, whereas 3886.50 is sensitive to β branching. Although no P4237.50 mutants produced dramatic EC50 shifts, cells injected with appropriate mRNA and tRNA for P4237.50L and P4237.50Lah mutations gave no response in our assay, suggesting this site is especially sensitive to steric bulk. Supporting this notion, the P4237.50 side chain points directly at TM1 in the D3 receptor crystal structure, while P1694.59 and P3886.50 are directed more toward the lipid bilayer.17 P2015.50. The 2015.50 site shows a qualitatively distinct pattern from the other prolines considered here. As before, R-hydroxy residues are less perturbing than their R-amino analogues (Figure 3, Table 1). Unlike the other proline sites, however, no hydroxy acid mutation to P2015.50 yields a wild-type EC50. P2015.50Aah and P2015.50Vah are loss of function by 4- and 8-fold, respectively (we were unable to characterize the P2015.50Lah mutant). Interestingly, N-Me-Ala is minimally perturbing, demonstrating

perhaps even gain of function, and the proline analogues Pip and Aze are wild-type (Table 2). Taken together, these data suggest that simply deleting the hydrogen bonding ability of the backbone NH is not sufficient to produce a maximally functional receptor. A substituent on the N is also necessary and can be either a ring (Pro, Pip, Aze) or a methyl (N-Me-Ala). Also in contrast to the TM4, 6, and 7 sites, Gly is highly disruptive at 2015.50, more so than Ala (Figure 3, Table 1). Together with the fact that no hydroxy acid tested gave a wildtype EC50 at this site, these findings suggest a more specific need for proline here, not merely a generic requirement for a helixbreaking residue. Phenotypes Correlate with Helix Distortions. In addition to sharing a functional phenotype, the TM4, 6, and 7 prolines all seem to play a similar structural role (Figure 1). In various structures of GPCRs, TM4, 6, and 7 all display a significant kink associated with the proline.17
21 Movements of helices 6 and 7 are considered to be critical to receptor activation, with the kinks playing a prominent role.16 Indeed, in a recent crystal structure thought to represent an active conformation of the β2 adrenergic receptor, displacement of TM6 is accomplished by a slight “unwinding” of the helical turn preceding the proline at position 6.50.36 The movement originates at residue 6.46, the would-be backbone hydrogen bond acceptor to position 6.50, potentially providing a direct link between proline 6.50's lack of hydrogen bond donor ability and functionally important helix flexibility. The 2015.50 site gave a unique functional phenotype, and indeed it is clear from Figure 1 that the structural perturbation associated with the TM5 proline is distinct from what is seen in helices 2, 4, 6, and 7. Instead of a prominent kink, helix 5 has a bulge. It has been proposed that this bulge is functionally significant, as residues in the bulge directly contact ligands in the agonist binding site. Specifically, the side chain of S1975.46, which is i
4 relative to P2015.50 and located at the center of the bulge, is thought to contribute a hydrogen bond that is important to agonist binding.37,38 Evidently, the steric effect of proline’s N-substitution is key to producing this structural bulge, consistent with the functional requirement for N-substitution we found at this site. Conclusions. We have probed five conserved proline sites located in the transmembrane region of the D2 receptor. Transmembrane prolines are not uncommon and are frequently considered to play important functional roles by introducing structural perturbations and/or increased conformational flexibility to the helix.4,15 In all functionally important transmembrane proline sites we have investigated to date, i.e., the TM4, 5, 6, and 7 prolines in this study and the M1 transmembrane proline of two different ligandgated ion channels, we find that lack of a backbone hydrogen bond donor is important for function.25,26 A general phenotype has emerged in which loss of function caused by mutations to conventional amino acids is mitigated by the corresponding R-hydroxy acids. In contrast to this transmembrane proline phenotype, R-hydroxy residues produced nonfunctional human 5-HT3 receptors when replacing a proline that appears to undergo cis
trans isomerization.27 This proline lies not in an R-helix, but in a loop/turn region, where cis
trans isomerization seems much more plausible. We do observe variations on the general transmembrane proline phenotype. Unlike the D2 receptor’s TM4, 6, and 7 prolines, the requirement for lacking a backbone hydrogen bond donor is absolute at the ion channel M1 proline sites; conventional amino acids, including glycine, produced completely nonfunctional channels.25,26 The TM5 proline of the D2 receptor 1066

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ACS Chemical Biology additionally requires N-substitution for fully wild-type function. Taken together, these studies establish the power of unnatural amino acid methodology for dissecting out the various consequences of the proline residue's unique structural features.

’ METHODS Molecular Biology. In these experiments, the cDNA for GIRK1 and GIRK4 was in pBSMXT plasmids and for the D2 receptor (human long form) was in the pGEMhe plasmid. Site-directed mutagenesis was performed using the Stratagene QuikChange protocol to generate the appropriate codon. For unnatural amino acid mutants and conventional mutants generated by nonsense suppression, the site of interest was mutated to the TAG stop codon. Plasmids were linearized with the appropriate restriction enzymes (the GIRK plasmids with SalI and the D2 receptor with NheI or SbfI). Receptor mRNA was prepared by in vitro runoff transcription using the Ambion T7 mMessage mMachine kit and GIRK1 and GIRK4 mRNA was prepared with the T3 kit. Hydroxy or amino acids, all commercially available, were appended to the dinucleotide dCA and enzymatically ligated to truncated 74mer THG73 tRNA as previously described.39,40 The 74mer tRNA was prepared using the Ambion T7MEGAshortscript kit by transcription from a DNA oligonucleotide template with its 50 terminal two nucleotides bearing methoxy groups at the ribose C20 position, as described in the literature to enhance RNA transcript homogeneity.41 Crude tRNAamino acid or tRNA-hydroxy acid product was used without desalting, and the product was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry on a 3-hydroxypicolinic acid matrix. Deprotection of the NVOC group on the tRNA-amino acids or NB group on tRNA-Aah was carried out by 5-min photolysis on a 1 kW xenon lamp with WG-335 and UG-11 filters immediately prior to injection. tRNA-Vah and -Lah were unprotected and were injected directly. Oocyte Preparation and RNA Injection. Stage V
VI oocytes of Xenopus laevis were harvested and injected with RNAs as described previously.39 For nonsense suppression experiments, each cell was injected with 15 ng each of GIRK1 and GIRK4 mRNA approximately 64 h before recording and then 4
30 ng of receptor mRNA and approximately 25
60 ng of appropriate tRNA approximately 48 h before recording. Mutants yielding small responses required a second injection of receptor mRNA and appropriate tRNA 24 h before recording. For wild-type experiments, each cell received a single injection of 0.16 ng of receptor mRNA and 10 ng each of GIRK1 and GIRK4 mRNA approximately 48 h before recording. Conventional amino acid mutants (except those generated by nonsense suppression) were prepared identically, except 1 ng of receptor mRNA was required. Small responses were obtained for P4237.50G, which required 10 ng of receptor mRNA. Injection volumes for each injection session were 25
75 nL per cell. As a negative control for suppression experiments at each site, unacylated full length tRNA was co-injected with mRNA in the same manner as charged tRNA. These experiments yielded negligible responses for all sites. Wild-type recovery conditions (injecting tRNA-Pro and appropriate mRNA) were injected alongside mutant nonsense suppression conditions to control for data variability. Electrophysiology. Oocyte recordings were made in two-electrode voltage clamp mode using the OpusXpress 6000A (Axon Instruments). Recording buffers were ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 1.8 mM CaCl2, pH 7.5) and high K+ ringer (96 mM NaCl, 24 mM KCl, 1 mM MgCl2, 5 mM HEPES, 1.8 mM CaCl2, pH 7.5). Solution flow rates were 2 mL min
1 and drug application flow rates were 2.5 mL min
1. Initial holding potential was
60 mV. Data were sampled at 125 Hz and filtered at 50 Hz. Cells were subjected to a ND96 pre-wash for 10 s, a high K+ application for 50 s to establish basal currents, and dopamine application in high K+

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ringer for 25 s, followed by high K+ and ND96 washings for 45 and 90 s in duration, respectively. Dopamine-induced currents were measured over the basal K+ current as described previously.31 Dopamine (SigmaAldrich) solutions in high K+ ringer were prepared immediately before recording by dilutions from a 1 M stock in ddi water. Dose
response data were obtained for a minimum of eight concentrations of dopamine, for a minimum of two cell batches, and for a minimum of 8 cells total. Dose
response relations were fitted to the Hill equation, Inorm = 1/(1 + (EC50/A))nH, where Inorm is the normalized current peak at [dopamine] = A, EC50 is the concentration of agonist that elicits a halfmaximum response, and nH is the Hill coefficient. EC50 values were obtained by averaging the Inorm values for each cell at a given dose and fitting those averaged Inorm data to the Hill equation.

’ ASSOCIATED CONTENT

bS

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Email: [email protected].

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